Performance-enhanced protease variants II

ABSTRACT

An amino acid sequence may have at least 70% sequence identity to the amino acid sequence identified in SEQ ID No. 1 over its entire length, and (a) amino acid substitutions at the positions corresponding to the positions 9 and 271 in each case based on the numbering according to SEQ ID No. 1, and (b) an amino acid substitution on at least one of the positions corresponding to the positions 29, 48, 101, 30, 131, 133, 144, 224 or 252, in each case based on the numbering according to SEQ ID No. 1. Such proteases are particularly suitable for having an improved cleaning performance.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a national stage entry according to 35 U.S.C.§ 371 of PCT application No.: PCT/EP2018/073880 filed on Sep. 5, 2018;which claims priority to German application No.: 10 2017 215 631.7 filedon Sep. 5, 2017, as well as claims priority to German application No.:10 2018 208 777.6 filed on Jun. 5, 2018; all of which are incorporatedherein by reference in their entirety and for all purposes.

REFERENCE TO A SEQUENCE LISTING SUBMITTED VIA EFS-WEB

The content of the ASCII text file of the sequence listing named“P75336US_sequencelisting_ST25” which is 58 kb in size was created onSep. 5, 2017 and electronically submitted via EFS-Web herewith. Thesequence listing is incorporated herein by reference in its entirety.

TECHNICAL FIELD

Proteases from Bacillus pumilus, the amino acid sequences of which havebeen altered to give them a better storage stability, in particular withregard to the use in washing and cleaning agents, and also relates tothe nucleic acids coding for said proteases and to the productionthereof.

BACKGROUND

Proteases are some of the technically most important enzymes. They arethe longest established enzymes for washing and cleaning agents, and arecontained in virtually all modern, effective washing and cleaningagents. They bring about the decomposition of protein-containing stainson the item to be cleaned. Of these, in turn, proteases of thesubtilisin type (subtilases, subtilopeptidases, EC 3.4.21.62) areparticularly important, which are serine proteases due to thecatalytically active amino acids. They act as non-specificendopeptidases and hydrolyze any acid amide bonds that are insidepeptides or proteins. Their optimum pH is usually in the distinctlyalkaline range. The article “Subtilases: Subtilisin-like Proteases” byR. Siezen, pages 75-95 in “Subtilisin enzymes,” published by R. Bott andC. Betzel, New York, 1996, gives an overview of this family, forexample. Subtilases are naturally formed from microorganisms. Inparticular, the subtilisins formed and secreted by Bacillus species arethe most significant group of subtilases.

Examples of the subtilisin proteases used in washing and cleaning agentsare the subtilisins BPN′ and Carlsberg, the protease PB92, thesubtilisins 147 and 309, the protease from Bacillus lentus, inparticular from Bacillus lentus DSM 5483, subtilisin DY and the enzymesthermitase, proteinase K and the proteases TW3 and TW7, which can beclassified as subtilases but no longer as subtilisins in the narrowersense, and variants of said proteases having an amino acid sequence thathas been altered with respect to the starting protease. Proteases arealtered in a targeted manner or randomly using methods known from theprior art, and are thus optimized for use in washing and cleaningagents, for example. This includes point, deletion or insertionmutagenesis, or fusion with other proteins or protein parts.Appropriately optimized variants are therefore known for the majority ofproteases known from the prior art.

European patent application EP2016175A1 discloses, for example, aprotease from Bacillus pumilus intended for washing and cleaning agents.In general, only selected proteases are suitable for use in liquid,surfactant-containing preparations in any case. Many proteases do notexhibit sufficient catalytic performance in such preparations. For theuse of proteases in cleaning agents, therefore, a high catalyticactivity under conditions as they are during a wash cycle and a highstorage stability is particularly desirable.

Consequently, protease and surfactant-containing liquid formulationsfrom the prior art are disadvantageous in that the proteases contained,under standard washing conditions (e.g. in a temperature range of from20° C. to 40° C.), do not have satisfactory proteolytic activity or arenot storage-stable and the formulations therefore do not exhibit optimalcleaning performance on protease-sensitive stains.

SUMMARY

Surprisingly, it has now been found that a protease from Bacilluspumilus or a sufficiently similar protease (based on the sequenceidentity) that has amino acid substitutions (a) at the positionscorresponding to positions 9 and 271, in each case based on thenumbering according to SEQ ID NO:1, and (b) at at least one of thepositions corresponding to positions 29, 48, 101, 130, 131, 133, 144,224 or 252, in each case based on the numbering according to SEQ IDNO:1, is improved in terms of storage stability compared with thewild-type form and/or reference mutants and is therefore particularlysuitable for use in washing or cleaning agents.

In a first aspect, a protease may include an amino acid sequence whichhas at least 70% sequence identity with the amino acid sequence given inSEQ ID NO:1 over its entire length and (i) has amino acid substitutions,such as the amino acid substitutions 9T, 9H, 9S or 9A and 271E, at thepositions corresponding to positions 9 and 271 according to SEQ ID NO:1,and (ii) has an amino acid substitution at at least one of the positionscorresponding to positions 29, 48, 101, 130, 131, 133, 144, 224 or 252,in each case based on the numbering according to SEQ ID NO:1.

In one embodiment of this first aspect, a protease may include an aminoacid sequence which has at least 70% sequence identity with the aminoacid sequence given in SEQ ID NO:1 over its entire length and has, ineach case based on the numbering according to SEQ ID NO:1:

-   -   (a) amino acid substitutions, in particular the amino acid        substitutions 9T, 130D, 144K, 252T and 271E, at the positions        corresponding to positions 9, 130, 144, 252 and 271; as well as        optionally    -   (b1) the amino acid substitution 166M, 166Q or 166A at the        position corresponding to position 166; and/or    -   (b2) at least one further amino acid substitution at at least        one of the positions corresponding to positions 62, 133, 149,        156, 172 or 217.

In a second aspect, a protease may include an amino acid sequence whichhas at least 70% sequence identity with the amino acid sequence given inSEQ ID NO:1 over its entire length and, based on the numbering accordingto SEQ ID NO:1, has at least one amino acid substitution at at least oneof the positions corresponding to positions 62, 149, 156, 166, 172 or217, the amino acid substitution at the position corresponding toposition 166 being selected from 166M, 166Q and 166A, and the amino acidsubstitution at the position corresponding to position 62 being selectedfrom 62S.

In a further aspect, a protease may have an amino acid sequence whichhas at least 70% sequence identity with the amino acid sequence given inSEQ ID NO:1 over its entire length and which comprises at least oneamino acid substitution selected from the group consisting of N130D,N130S, N130H, G131N, G131D, G131K, T133K, T133Y, N144L, N144A, S224A, orN252S, in each case based on the numbering according to SEQ ID NO:1. Insuch an embodiment, the protease has either no substitutions atpositions 9 and 271, and optionally also no substitution at position216, or it has a substitution at one of positions 9 and 271, andoptionally also a substitution at position 216.

In various embodiments of the aspects described above, the sequenceidentity with SEQ ID NO:1 may be 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%,91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%,97.5%, 98%, 98.5%, or 98.8%.

A method for producing a protease, may include the substitution of aminoacids in a starting protease which has at least 70% sequence identitywith the amino acid sequence given in SEQ ID NO:1 over its entire length(i) at the positions corresponding to positions 9 and 271 in SEQ IDNO:1, such that the protease comprises the amino acid substitutions 9T,9H, 9S or 9A, in particular 9T and 271E, at the positions, and (ii) atat least one position corresponding to position 29, 48, 101, 130, 131,133, 144, 224 or 252 in SEQ ID NO:1, such that the protease has at leastone of the amino acid substitutions 29G, 48V, 101E, 130D, 130S, 130H,131S, 131N, 131D, 131K, 133K, 133R, 133Y, 144K, 144L, 144A, 224A, 224T,252T or 252S. The protease that can be obtained by this method has atleast 70% sequence identity with the amino acid sequence given in SEQ IDNO:1 over its entire length.

In various embodiments of this aspect, a method for producing a proteaseas defined above, may include the substitution of amino acids in astarting protease which has at least 70% sequence identity with theamino acid sequence given in SEQ ID NO:1 over its entire length (i) atthe positions corresponding to positions 9, 130, 144, 252 and 271 in SEQID NO:1, such that the protease comprises amino acid substitutions, inparticular the amino acid substitutions 9T, 130D, 144K, 252T and 271E,at the positions, and optionally (ii) at the position corresponding toposition 166 in SEQ ID NO:1, such that the protease has at least one ofthe amino acid substitutions 166M, 166Q or 166A and/or (iii) has atleast one further amino acid substitution at at least one of thepositions corresponding to positions 62, 133, 149, 156, 172 or 217 inSEQ ID NO:1. The protease that can be obtained by this method has atleast 70% sequence identity with the amino acid sequence given in SEQ IDNO:1 over its entire length.

DETAILED DESCRIPTION

A protease within the meaning of the present patent applicationtherefore comprises both the protease as such and a protease produced bya method. All statements regarding the protease therefore relate both tothe protease as such and to the proteases produced by means ofcorresponding methods.

Further aspects relate to the nucleic acids coding for these proteases,to non-human host cells containing proteases or nucleic acids, and toagents comprising proteases, in particular washing and cleaning agents,to washing and cleaning methods, and to uses of the proteases in washingor cleaning agents in order to remove protein-containing stains.

“At least one,” as used herein, means one or more, i.e. 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14 or more.

When the protease is defined herein such that it includes “the aminoacid substitutions 9T, 9H, 9S or 9A,” this means that position 9 ismutated to either T, H, S or A. Thus, the phrase whereby the proteasecomprises “the amino acid substitutions 9T, 9H, 9S or 9A and 271E andoptionally 216C” means that position 9 is mutated to either T, H, S orA, position 271 is mutated to E and position 216 is optionally mutatedto C.

A surprising finding by the inventors relates to amino acidsubstitutions at the positions corresponding to positions 9 and 271, ineach case based on the numbering according to SEQ ID NO:1, such that theprotease comprises the amino acid substitutions 9T, 9H, 9S or 9A and271E at the positions, and an amino acid substitution at at least one ofthe positions corresponding to positions 29, 48, 101, 130, 131, 133,144, 224 or 252 of the protease from Bacillus pumilus according to SEQID NO:1, in a protease comprising an amino acid sequence that is atleast 70% identical to the amino acid sequence given in SEQ ID NO:1,such that the amino acids 29G, 48V, 101E, 130D, 130S, 130H, 131S, 131N,131D, 131K, 133K, 133R, 133Y, 144K, 144L, 144A, 224A, 224T, 252T or 252Sare present at at least one of the corresponding positions, results inimproved storage stability of this altered protease in washing andcleaning agents. It has further been found that this storage stabilityin proteases based on the sequence according to SEQ ID NO:1, which haveamino acid substitutions, in particular the amino acid substitutions 9T,130D, 144K, 252T and 271E, at the positions corresponding to positions9, 130, 144, 252 and 271; and optionally (b1) have the amino acidsubstitutions 166M, 166Q or 166A at the position corresponding toposition 166; and/or (b2) have at least one further amino acidsubstitution at at least one of the positions corresponding to positions62, 133, 149, 156, 172 or 217, is improved in a particularlyadvantageous manner. This is particularly surprising insofar as none ofthe above-mentioned amino acid substitutions has previously beenassociated with increased storage stability of the protease. Optionally,improved performance of this altered protease (proteolytic activityunder standard washing conditions) can additionally be brought about inwashing and cleaning agents.

In various embodiments of the protease, the protease has amino acidsubstitutions

-   -   (A) (i) at the positions corresponding to positions 9, 131 and        271, in each case based on the numbering according to SEQ ID        NO:1, and optionally (ii) at at least one of the positions        corresponding to positions 29, 48, 101, 130, 133, 144, 224 or        252, in each based on the numbering according to SEQ ID NO:1;    -   (B) (i) at the positions corresponding to positions 9, 133 and        271, in each case based on the numbering according to SEQ ID        NO:1, and optionally (ii) at at least one of the positions        corresponding to positions 29, 48, 101, 130, 131, 144, 224 or        252, in each based on the numbering according to SEQ ID NO:1;    -   (c) (i) at the positions corresponding to positions 9, 224 and        271, in each case based on the numbering according to SEQ ID        NO:1, and optionally (ii) at at least one of the positions        corresponding to positions 29, 48, 101, 130, 131, 133, 144 or        252, in each based on the numbering according to SEQ ID NO:1;    -   (D) (i) at the positions corresponding to positions 9, 130 and        271, in each case based on the numbering according to SEQ ID        NO:1, and optionally (ii) at at least one of the positions        corresponding to positions 29, 48, 101, 131, 133, 144, 224 or        252, in each based on the numbering according to SEQ ID NO:1; or    -   (E) (i) at the positions corresponding to positions 9, 144 and        271, in each case based on the numbering according to SEQ ID        NO:1, and optionally (ii) at at least one of the positions        corresponding to positions 29, 48, 101, 130, 131, 133, 224 or        252, in each based on the numbering according to SEQ ID NO:1;    -   (F) (i) at the positions corresponding to positions 9, 252 and        271, in each case based on the numbering according to SEQ ID        NO:1, and optionally (ii) at at least one of the positions        corresponding to positions 29, 48, 101, 130, 131, 133, 144 or        224, in each based on the numbering according to SEQ ID NO:1;    -   (G) (i) at the positions corresponding to positions 9, 130, 133        and 271, in each case based on the numbering according to SEQ ID        NO:1, and optionally (ii) at at least one of the positions        corresponding to positions 29, 48, 101, 131, 144, 224 or 252, in        each based on the numbering according to SEQ ID NO:1;    -   (H) (i) at the positions corresponding to positions 9, 130, 144        and 271, in each case based on the numbering according to SEQ ID        NO:1, and optionally (ii) at at least one of the positions        corresponding to positions 29, 48, 101, 131, 133, 224 or 252, in        each based on the numbering according to SEQ ID NO:1;    -   (0) (i) at the positions corresponding to positions 9, 130, 252        and 271, in each case based on the numbering according to SEQ ID        NO:1, and optionally (ii) at at least one of the positions        corresponding to positions 29, 48, 101, 131, 133, 144 or 224, in        each based on the numbering according to SEQ ID NO:1;    -   (J) (i) at the positions corresponding to positions 9, 133, 144        and 271, in each case based on the numbering according to SEQ ID        NO:1, and optionally (ii) at at least one of the positions        corresponding to positions 29, 48, 101, 130, 131, 224 or 252, in        each based on the numbering according to SEQ ID NO:1;    -   (K) (i) at the positions corresponding to positions 9, 133, 252        and 271, in each case based on the numbering according to SEQ ID        NO:1, and optionally (ii) at at least one of the positions        corresponding to positions 29, 48, 101, 130, 131, 144 or 224, in        each based on the numbering according to SEQ ID NO:1;    -   (L) (i) at the positions corresponding to positions 9, 144, 252        and 271, in each case based on the numbering according to SEQ ID        NO:1, and optionally (ii) at at least one of the positions        corresponding to positions 29, 48, 101, 130, 131, 133 or 224, in        each based on the numbering according to SEQ ID NO:1;    -   (M) (i) at the positions corresponding to positions 9, 130, 144,        252 and 271, in each case based on the numbering according to        SEQ ID NO:1, and optionally (ii) at at least one of the        positions corresponding to positions 29, 48, 101, 131, 133 or        224, in each based on the numbering according to SEQ ID NO:1;    -   (N) (i) at the positions corresponding to positions 9, 133, 144,        252 and 271, in each case based on the numbering according to        SEQ ID NO:1, and optionally (ii) at at least one of the        positions corresponding to positions 29, 48, 101, 130, 131 or        224, in each based on the numbering according to SEQ ID NO:1;    -   (O) (i) at the positions corresponding to positions 9, 130, 133,        252 and 271, in each case based on the numbering according to        SEQ ID NO:1, and optionally (ii) at at least one of the        positions corresponding to positions 29, 48, 101, 131, 144 or        224, in each based on the numbering according to SEQ ID NO:1;    -   (P) (i) at the positions corresponding to positions 9, 130, 133,        144 and 271, in each case based on the numbering according to        SEQ ID NO:1, and optionally (ii) at at least one of the        positions corresponding to positions 29, 48, 101, 131, 224 or        252, in each based on the numbering according to SEQ ID NO:1;    -   (Q) (i) at the positions corresponding to positions 9, 130, 133,        144, 252 and 271, in each case based on the numbering according        to SEQ ID NO:1, and optionally (ii) at at least one of the        positions corresponding to positions 29, 48, 101, 131 or 224, in        each based on the numbering according to SEQ ID NO:1; or    -   (R) (i) has amino acid substitutions, in particular the amino        acid substitutions 9T, 130D, 144K, 252T and 271E, at the        positions corresponding to positions 9, 130, 144, 252 and 271;        and (ii) has one of the amino acid substitutions 62S, 149l,        156G, 156Q, 166M, 166Q, 166A, 172P, 172G or 217M at one of the        positions corresponding to positions 62, 149, 156, 166, 172 or        217.

In further embodiments, proteases are those having amino acidsubstitutions at the positions:

9 and 271 and at least one of 131, 133, 224, 130 and 144, and optionallyat least one of 29, 48, 101 and 252;

9+271+131+224 and optionally at least one of 29, 48, 101, 130, 133, 144or 252;

9+271+131+130 and optionally at least one of 29, 48, 101, 130, 144, 224or 252;

9+271+131+130+224 and optionally at least one of 29, 48, 101, 130, 144or 252; or

9+271+131+133+224+130 and optionally at least one of 29, 48, 101, 144 or252;

Here, the corresponding exchanges are in particular those mentionedabove, i.e. 9T, 9H, 9S, 9A, 29G, 48V, 101E, 130D, 130S, 130H, 131D,131N, 131S, 131K, 133K, 133R, 133Y, 144K, 144L, 144A, 224A, 224T, 252S,252T and/or 271E.

In various embodiments, the protease has

-   -   (1) amino acid substitutions, in particular the amino acid        substitutions 9T, 130D, 144K, 252T and 271E, at the positions        corresponding to positions 9, 130, 144, 252 and 271; and    -   (2) one of the amino acid substitutions 62S, 1491, 156G, 156Q,        166M, 166Q, 166A, 172P, 172G or 217M at one of the positions        corresponding to positions 62, 149, 156, 166, 172 or 217;

In embodiments of the protease, the protease has substitutions selectedfrom the amino acid substitutions 149l, 156G, 156Q, 172P, 172G and 217Mat the positions corresponding to positions 149, 156, 172 or 217.

In various embodiments, the protease has amino acid substitutions, inparticular the amino acid substitutions 9T, 130D, 144K, 252T and 271E,at the positions corresponding to positions 9, 130, 144, 252 and 271;and has at least one, for example 1, 2, 3, 4, 5 or 6, for example 1, 2,3 or 4, further amino acid substitutions at one or more of the positionscorresponding to positions 62, 149, 156, 166, 172 or 217, these beingselected from: 62S, 1491, 156G, 156Q, 166M, 166Q, 166A, 172P, 172G or217M. Such proteases are disclosed for example as mutants 7-12 inexample. Particularly suitable are those proteases which have an aminoacid substitution, in particular the amino acid substitution 271E, atthe position corresponding to position 271; and have the amino acidsubstitutions 9T, 130D, 144K and 252T at the positions corresponding topositions 9, 130, 144 and 252; and at least one, for example 1, 2 or 3,further amino acid substitution(s) at one or more of the positionscorresponding to positions 62, 166 or 217, these being selected from:62S, 166Q and 217M.

In further embodiments, the protease has amino acid substitutions at thepositions (in the numbering according to SEQ ID NO:1):

-   9+271+29-   9+271+48-   9+271+101-   9+271+130-   9+271+131-   9+271+133-   9+271+144-   9+271+224-   9+271+252-   9+271+29+48-   9+271+29+101-   9+271+29+130-   9+271+29+131-   9+271+29+133-   9+271+29+144-   9+271+29+224-   9+271+29+252-   9+271+48+101-   9+271+48+130-   9+271+48+131-   9+271+48+133-   9+271+48+144-   9+271+48+224-   9+271+48+252-   9+271+101+130-   9+271+101+131-   9+271+101+133-   9+271+101+144-   9+271+101+224-   9+271+101+252-   9+271+130+131-   9+271+130+133-   9+271+130+144-   9+271+130+224-   9+271+130+252-   9+271+131+133-   9+271+131+144-   9+271+131+224-   9+271+131+252-   9+271+133+144-   9+271+133+224-   9+271+133+252-   9+271+144+224-   9+271+144+252-   9+271+224+252-   9+271+29+48+101-   9+271+29+48+130-   9+271+29+48+131-   9+271+29+48+133-   9+271+29+48+144-   9+271+29+48+224-   9+271+29+48+252-   9+271+29+101+130-   9+271+29+101+131-   9+271+29+101+133-   9+271+29+101+144-   9+271+29+101+224-   9+271+29+101+252-   9+271+29+130+131-   9+271+29+130+133-   9+271+29+130+144-   9+271+29+130+224-   9+271+29+130+252-   9+271+29+131+133-   9+271+29+131+144-   9+271+29+131+224-   9+271+29+131+252-   9+271+29+133+144-   9+271+29+133+224-   9+271+29+133+252-   9+271+29+144+224-   9+271+29+144+252-   9+271+29+224+252-   9+271+48+101+130-   9+271+48+101+131-   9+271+48+101+133-   9+271+48+101+144-   9+271+48+101+224-   9+271+48+101+252-   9+271+48+130+131-   9+271+48+130+133-   9+271+48+130+144-   9+271+48+130+224-   9+271+48+130+252-   9+271+48+131+133-   9+271+48+131+144-   9+271+48+131+224-   9+271+48+131+252-   9+271+48+133+144-   9+271+48+133+224-   9+271+48+133+252-   9+271+48+144+224-   9+271+48+144+252-   9+271+48+224+252-   9+271+101+130+131-   9+271+101+130+133-   9+271+101+130+144-   9+271+101+130+224-   9+271+101+130+252-   9+271+101+131+133-   9+271+101+131+144-   9+271+101+131+224-   9+271+101+131+252-   9+271+101+133+144-   9+271+101+133+224-   9+271+101+133+252-   9+271+101+144+224-   9+271+101+144+252-   9+271+101+224+252-   9+271+130+131+133-   9+271+130+131+144-   9+271+130+131+224-   9+271+130+131+252-   9+271+130+133+144-   9+271+130+133+224-   9+271+130+133+252-   9+271+130+144+224-   9+271+130+144+252-   9+271+130+224+252-   9+271+131+133+144-   9+271+131+133+224-   9+271+131+133+252-   9+271+131+144+224-   9+271+131+144+252-   9+271+131+224+252-   9+271+133+144+224-   9+271+133+144+252-   9+271+133+224+252-   9+271+144+224+252-   9+271+29+48+101+130-   9+271+29+48+101+131-   9+271+29+48+101+133-   9+271+29+48+101+144-   9+271+29+48+101+224-   9+271+29+48+101+252-   9+271+29+48+130+131-   9+271+29+48+130+133-   9+271+29+48+130+144-   9+271+29+48+130+224-   9+271+29+48+130+252-   9+271+29+48+131+133-   9+271+29+48+131+144-   9+271+29+48+131+224-   9+271+29+48+131+252-   9+271+29+48+133+144-   9+271+29+48+133+224-   9+271+29+48+133+252-   9+271+29+101+130+131-   9+271+29+101+130+133-   9+271+29+101+130+144-   9+271+29+101+130+224-   9+271+29+101+130+252-   9+271+29+101+131+133-   9+271+29+101+131+144-   9+271+29+101+131+224-   9+271+29+101+131+252-   9+271+29+101+133+144-   9+271+29+101+133+224-   9+271+29+101+133+252-   9+271+29+101+144+224-   9+271+29+101+144+252-   9+271+29+101+224+252-   9+271+29+130+131+133-   9+271+29+130+131+144-   9+271+29+130+131+224-   9+271+29+130+131+252-   9+271+29+130+133+144-   9+271+29+130+133+224-   9+271+29+130+133+252-   9+271+29+130+144+224-   9+271+29+130+144+252-   9+271+29+130+224+252-   9+271+29+131+133+144-   9+271+29+131+133+224-   9+271+29+131+133+252-   9+271+29+131+144+224-   9+271+29+131+144+252-   9+271+29+131+224+252-   9+271+29+133+144+224-   9+271+29+133+144+252-   9+271+29+133+224+252-   9+271+29+144+224+252-   9+271+48+101+130+131-   9+271+48+101+130+133-   9+271+48+101+130+144-   9+271+48+101+130+224-   9+271+48+101+130+252-   9+271+48+101+131+133-   9+271+48+101+131+144-   9+271+48+101+131+224-   9+271+48+101+131+252-   9+271+48+101+133+144-   9+271+48+101+133+224-   9+271+48+101+133+252-   9+271+48+101+144+224-   9+271+48+101+144+252-   9+271+48+101+224+252-   9+271+48+130+131+133-   9+271+48+130+131+144-   9+271+48+130+131+224-   9+271+48+130+131+252-   9+271+48+130+133+144-   9+271+48+130+133+224-   9+271+48+130+133+252-   9+271+48+130+144+224-   9+271+48+130+144+252-   9+271+48+130+224+252-   9+271+48+131+133+144-   9+271+48+131+133+224-   9+271+48+131+133+252-   9+271+48+131+144+224-   9+271+48+131+144+252-   9+271+48+131+224+252-   9+271+48+133+144+224-   9+271+48+133+144+252-   9+271+48+133+224+252-   9+271+48+144+224+252-   9+271+101+130+131+133-   9+271+101+130+131+144-   9+271+101+130+131+224-   9+271+101+130+131+252-   9+271+101+130+133+144-   9+271+101+130+133+224-   9+271+101+130+133+252-   9+271+101+130+144+224-   9+271+101+130+144+252-   9+271+101+130+224+252-   9+271+101+131+133+144-   9+271+101+131+133+224-   9+271+101+131+133+252-   9+271+101+131+144+224-   9+271+101+131+144+252-   9+271+101+131+224+252-   9+271+101+133+144+224-   9+271+101+133+144+252-   9+271+101+133+224+252-   9+271+101+144+224+252-   9+271+130+131+133+144-   9+271+130+131+133+224-   9+271+130+131+133+252-   9+271+130+131+144+224-   9+271+130+131+144+252-   9+271+130+131+224+252-   9+271+130+133+144+224-   9+271+130+133+144+252-   9+271+130+133+224+252-   9+271+130+144+224+252-   9+271+131+133+144+224-   9+271+131+133+144+252-   9+271+131+133+224+252-   9+271+131+144+224+252-   9+271+133+144+224+252

In various embodiments, the aforementioned variants do not have furthersubstitutions or have only one or more additional substitutions in thepositions from the group of positions of 29, 48, 101, 130, 131, 133,144, 224 and 252, if these have not yet been mentioned above. In furtherembodiments, in particular in all the embodiments described above, theprotease has at least one, for example 1, 2 or 3, additional amino acidsubstitutions at the positions corresponding to positions 62, 166 or217, based on the numbering according to SEQ ID NO:1. In variousembodiments, this additional amino acid substitution is a substitutionat position 217. In various other embodiments, this additionalsubstitution is one at position 62 and/or 166. The above-described aminoacid substitution may be selected from: 62S, 166Q and 217M.

Further embodiments relate to protease variants which have amino acidsubstitutions at the following positions (in the numbering according toSEQ ID NO:1):

-   9+130+144+252+271+62-   9+130+144+252+271+149-   9+130+144+252+271+156-   9+130+144+252+271+166-   9+130+144+252+271+172-   9+130+144+252+271+217-   9+130+144+252+271+62+149-   9+130+144+252+271+62+156-   9+130+144+252+271+62+166-   9+130+144+252+271+62+172-   9+130+144+252+271+62+217-   9+130+144+252+271+149+156-   9+130+144+252+271+149+166-   9+130+144+252+271+149+172-   9+130+144+252+271+149+217-   9+130+144+252+271+156+166-   9+130+144+252+271+156+172-   9+130+144+252+271+156+217-   9+130+144+252+271+166+172-   9+130+144+252+271+166+217-   9+130+144+252+271+172+217-   9+130+133+144+252+271+62-   9+130+133+144+252+271+149-   9+130+133+144+252+271+156-   9+130+133+144+252+271+166-   9+130+133+144+252+271+172-   9+130+133+144+252+271+217-   9+130+133+144+252+271+62+149-   9+130+133+144+252+271+62+156-   9+130+133+144+252+271+62+166-   9+130+133+144+252+271+62+172-   9+130+133+144+252+271+62+217-   9+130+133+144+252+271+149+156-   9+130+133+144+252+271+149+166-   9+130+133+144+252+271+149+172-   9+130+133+144+252+271+149+217-   9+130+133+144+252+271+156+166-   9+130+133+144+252+271+156+172-   9+130+133+144+252+271+156+217-   9+130+133+144+252+271+166+172-   9+130+133+144+252+271+166+217-   9+130+133+144+252+271+172+217

In various embodiments, the aforementioned variants do not have furthersubstitutions or have only one or more additional substitutions in thepositions from the group of positions of 62, 133, 149, 156, 166, 172 and217, if these have not yet been mentioned above.

In all the aforementioned variants, the corresponding exchanges are inparticular those mentioned above, i.e. 9T, 9H, 9S, 9A, 29G, 48V, 101E,130D, 130S, 130H, 131D, 131N, 131S, 131K, 133K, 133R, 133Y, 144K, 144L,144A, 224A, 224T, 252S, 252T and/or 271E. 9T, 130D, 133R/K, 144K, 252T/Sand/or 271E, in particular 9T, 130D, 133R/K, 144K, 252T and 271E or 9T,130D, 133K, 144K, 252T and 271E, are particularly suitable. A variantwhich has the substitutions 9T, 130D, 144K, 252T and 271E, andoptionally also 133R/K and/or 217M, is further possible.

In embodiments of the protease, i.e. in particular the variants listedabove, the protease has amino acid substitutions at the positionscorresponding to positions 62, 149, 156, 166, 172 and 217, these beingselected from: 62S, 149l, 156G, 156Q, 166M, 166Q, 166A, 172P, 172G or217M.

Such proteases are disclosed for example as mutants in example 1.Particularly suitable are those proteases which have an amino acidsubstitution, in particular the amino acid substitution 271E, at theposition corresponding to position 271; and have the amino acidsubstitutions 9T, 130D, 144K and 252T at the positions corresponding topositions 9, 130, 144 and 252; and at least one, for example 1, 2 or 3,further amino acid substitution(s) at one or more of the positionscorresponding to positions 62, 166 or 217, these being selected from:62S, 166Q and 217M.

In further embodiments, in particular in all the embodiments describedabove, the protease has an additional amino acid substitution at theposition corresponding to position 216, based on the numbering accordingto SEQ ID NO:1. This amino acid substitution may be the amino acidsubstitution S216C. In various embodiments, however, the applicationdescribed herein also relates to protease variants which do not have thesubstitution 216C, in particular do not have a substitution in theposition 216 in the numbering according to SEQ ID NO:1.

Furthermore, in various embodiments, the protease contains at least oneamino acid substitution selected from the group consisting of A29G,A48V, D101E, N130D, N130S, N130H, G131S, G131N, G131D, G131K, T133K,T133R, T133Y, N144K, N144L, N144A, S224A, S224T or N252S, in each casebased on the numbering according to SEQ ID NO:1. In yet furtherembodiments, the protease contains the amino acid substitutions (1) P9T,P9H, P9S or P9A and (2) Q271E and optionally also (3) S216C andadditionally one of the following amino acid substitution variants: (i)N130D, G131S, T133Y and S224A; (ii) N130S, G131S, T133Y, N144L andS224A; (iii) N130D, G131N, T133K and N144K; (iv) N130H, G131N, N144K andS224A; (v) A29G, N130D, G131N and T133R; (vi) A29G, D101E, N130S, G131S,S224T and N252S; (vii) N130H, G131S and S224A; (viii) N130D, G131S,T133K and S224A; (ix) A29G, D101E, N130D, G131K and S224A; (x) N130D,G131N T133K, N144L and N252S; (xi) G131S, N144K and S224T; (xii) D101E,N130D, G131S, T133Y, N144A and S224A; (xiii) A29G, D101E, N130S, S224Tand N252S; (xiv) A48V, G131S, T133R and S224A; or (xv) G131D, T133R andS224A, the numbering being based in each case on the numbering accordingto SEQ ID NO:1.

In a further embodiment, the protease has an amino acid sequenceaccording to SEQ ID Nos. 3-17 or 19-23 or 20-23.

The proteases have improved storage stability. They have increasedstability in washing or cleaning agents in comparison with a referencemutation variant of the protease (SEQ ID NO:18 and/or SEQ ID NO:2 and/orSEQ ID NO: 19), in particular when stored for 3 or more days, 4 or moredays, 7 or more days, 10 or more days, 12 or more days, 14 or more days,21 or more days or 28 or more days. Such stability-enhanced proteasesmake it possible, even after a prolonged storage time, to achieve goodwashing results on proteolytically sensitive stains in varioustemperature ranges, in particular in a temperature range of from 20° C.to 40° C.

In addition to increased storage stability, the proteases may also haveincreased catalytic activity in washing or cleaning agents. In variousembodiments, the proteases may have a proteolytic activity which, basedon the wild type (SEQ ID NO:1), is at least 101%, such as at least 102%or more. In various embodiments, the proteases have a proteolyticactivity which, based on a reference mutation variant of the protease(SEQ ID NO:1 and/SEQ ID NO:2 and/or SEQ ID NO: 19), is at least 110%, atleast 115%, at least 120%, at least 125%, at least 130%, at least 135%,at least 140%, at least 145%, at least 150%, at least 155% or at least160%. Such performance-enhanced proteases allow improved washing resultson proteolytically sensitive stains in various temperature ranges, inparticular in a temperature range of from 20° C. to 40° C.

The proteases exhibit enzymatic activity, i.e. they are capable ofhydrolyzing peptides and proteins, in particular in a washing orcleaning agent. A protease is therefore an enzyme which catalyzes thehydrolysis of amide/peptide bonds in protein/peptide substrates and isthus able to cleave proteins or peptides. Furthermore, a protease is amature protease, i.e. the catalytically active molecule without signalpeptide(s) and/or propeptide(s). Unless stated otherwise, the sequencesgiven also each refer to mature (processed) enzymes.

In various embodiments, the protease is a free enzyme. This means thatthe protease can act directly with all the components of an agent and,if the agent is a liquid agent, that the protease is in direct contactwith the solvent of the agent (e.g. water). In other embodiments, anagent may contain proteases that form an interaction complex with othermolecules or that contain a “coating.” In this case, an individualprotease molecule or multiple protease molecules may be separated fromthe other constituents of the agent by a surrounding structure. Such aseparating structure may arise from, but is not limited to, vesiclessuch as a micelle or a liposome. The surrounding structure may also be avirus particle, a bacterial cell or a eukaryotic cell. In variousembodiments, an agent may include cells of Bacillus pumilus or Bacillussubtilis which express the proteases, or cell culture supernatants ofsuch cells.

In a further embodiment, the protease comprises an amino acid sequencewhich, over its entire length, is at least 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%,96%, 96.5%, 97%, 97.5%, 98%, 98.5% and 98.8% identical to the amino acidsequence given in SEQ ID NO:1, and has the amino acid substitutionsgiven above in each case based on the numbering according to SEQ IDNO:1. In the context of the present invention, the feature whereby aprotease has the given substitutions means that it contains one (of thegiven) substitution(s) at the relevant position, i.e. at least the givenpositions are not otherwise mutated or deleted, for example byfragmenting of the protease. In various embodiments, it has amino acidsubstitutions (a) at the positions corresponding to positions 9 and 271,such that the amino acid substitutions are P9T, P9H, P9S or P9A andQ271E, and (b) has one or more of the amino acid substitutions 29G, 48V,101E, 130D, 130S, 130H, 131S, 131N, 131D, 131K, 133K, 133R, 133Y, 144K,144L, 144A, 224A, 224T or 252S at at least one of the positionscorresponding to positions 29, 48, 101, 130, 131, 133, 144, 224 or 252in the numbering according to SEQ ID NO:1. The amino acid sequences ofsuch proteases which are suitable are given in SEQ ID Nos: 3-17 and19-23. In various embodiments, the proteases described herein, with theexception of the explicitly mentioned substitutions, have the sequenceof SEQ ID NO:1, i.e. apart from the substituted positions, they are 100%identical to the sequence according to SEQ ID NO:1.

The identity of nucleic acid or amino acid sequences is determined by asequence comparison. This sequence comparison is based on the BLASTalgorithm established and commonly used in the prior art (cf. forexample Altschul et al. (1990): “Basic local alignment search tool,” J.Mol. Biol. 215: 403-410, and Altschul et al. (1997): “Gapped BLAST andPSI-BLAST: a new generation of protein database search programs,”Nucleic Acids Res. 25:3389-3402) and in principle occurs by associatingsimilar sequences of nucleotides or amino acids in the nucleic acid oramino acid sequences. A tabular association of the positions concernedis referred to as alignment. Another algorithm available in the priorart is the FASTA algorithm. Sequence comparisons (alignments), inparticular multiple sequence comparisons, are created using computerprograms. The Clustal series (cf. for example, Chenna et al. (2003):“Multiple sequence alignment with the Clustal series of programs,”Nucleic Acid Res. 31:3497-3500), T-Coffee (cf. for example, Notredame etal. (2000): “T-Coffee: A novel method for multiple sequence alignments,”J. Mol. Biol. 302: 205-217) or programs based on these programs oralgorithms are frequently used, for example. Sequence comparisons(alignments) using the computer program Vector NTI® Suite 10.3(Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, Calif., USA)with the predetermined, default parameters, and the AlignX module ofwhich for sequence comparisons is based on ClustalW, are also possible.Unless stated otherwise, the sequence identity given herein isdetermined by the BLAST algorithm.

Such a comparison also allows a statement regarding the similarity ofthe compared sequences. It is usually given in percent identity, i.e.the proportion of identical nucleotides or amino acid residues in saidsequences or in an alignment of corresponding positions. The broaderconcept of homology takes conserved amino acid exchanges into account inthe case of amino acid sequences, i.e. amino acids having similarchemical activity, since they usually perform similar chemicalactivities within the protein. Therefore, the similarity of the comparedsequences may also be stated as percent homology or percent similarity.Identity and/or homology information can be provided regarding wholepolypeptides or genes or only regarding individual regions. Homologousor identical regions of different nucleic acid or amino acid sequencesare therefore defined by matches in the sequences. Such regions oftenhave identical functions. They can be small and comprise only a fewnucleotides or amino acids. Often, such small regions perform essentialfunctions for the overall activity of the protein. It may therefore beexpedient to relate sequence matches only to individual, optionallysmall regions. Unless stated otherwise, however, identity or homologyinformation in the present application relates to the entire length ofthe particular nucleic acid or amino acid sequence indicated.

In the context, the indication that an amino acid position correspondsto a numerically designated position in SEQ ID NO:1 therefore means thatthe corresponding position is associated with the numerically designatedposition in SEQ ID NO:1 in an alignment as defined above.

In a further embodiment, the protease is characterized in that thecleaning performance thereof (after storage, e.g. over 4 weeks) is notsignificantly reduced compared with that of a protease comprising anamino acid sequence that corresponds to the amino acid sequence given inSEQ ID NO:18 and/or SEQ ID NO:2 and/or SEQ ID NO:19, i.e. has at least80% of the reference washing performance, such as at least 100%, or atleast 110% or more. The cleaning performance can be determined in awashing system containing a washing agent in a dosage between 4.5 and7.0 grams per liter of washing liquor and the protease, the proteases tobe compared being used in the same concentration (based on activeprotein) and the cleaning performance with respect to a stain on cottonbeing determined by measuring the degree of cleaning of the washedtextiles. For example, the washing process can take place for 60 minutesat a temperature of 40° C. and the water can have a water hardnessbetween 15.5 and 16.5° (German hardness). The concentration of theprotease in the washing agent intended for this washing system is 0.001to 0.1 wt. %, such as 0.01 to 0.06 wt. % based on active, purifiedprotein.

A liquid reference washing agent for such a washing system may becomposed as follows (all values in wt. %): 4.4% alkyl benzene sulfonicacid, 5.6% further anionic surfactants, 2.4% C12-C18 Na salts of fattyacids (soaps), 4.4% non-ionic surfactants, 0.2% phosphonates, 1.4%citric acid, 0.95% NaOH, 0.01% defoamer, 2% glycerol, 0.08%preservatives, 1% ethanol, and the remainder being demineralized water.In a non-limiting embodiment, the dosage of the liquid washing agent isbetween 4.5 and 6.0 grams per liter of washing liquor, for example 4.7,4.9 or 5.9 grams per liter of washing liquor. Washing in a pH rangebetween pH 7 and pH 10.5, such as between pH 7.5 and pH 8.5, ispossible.

In the context, the cleaning performance is determined for example at20° C. or 40° C. using a liquid washing agent as specified above, thewashing process being carried out for 60 minutes at 600 rpm.

The degree of whiteness, i.e. the lightening of stains, as a measure ofthe cleaning performance is determined by optical measuring methods,such as photometrically. A suitable device for this purpose is forexample the Minolta CM508d spectrometer. Usually, the devices used forthe measurement are calibrated beforehand with a white standard, such asa supplied white standard.

The activity-equivalent use of the relevant protease ensures that therespective enzymatic properties, for example the cleaning performance oncertain stains, are compared even if the ratio of active substance tototal protein (the values of the specific activity) significantlydiffers. In general, a low specific activity can be compensated for byadding a larger amount of protein.

Otherwise, methods for determining protease activity are well known to,and routinely used by, a person skilled in the art of enzyme technology.For example, such methods are disclosed in Tenside, vol. 7 (1970), p.125-132. Alternatively, the protease activity can be determined by therelease of the chromophore para-nitroaniline (pNA) from the substratesuc-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (AAPF). The protease cleavesthe substrate and releases pNA. The release of the pNA causes anincrease in absorbance at 410 nm, the temporal progression of which is ameasure of the enzymatic activity (cf. Del Mar et al., 1979). Themeasurement is carried out at a temperature of 25° C., a pH of 8.6, anda wavelength of 410 nm. The measuring time is 5 min and the measuringinterval is 20 s to 60 s. The protease activity is usually indicated inprotease units (PE). Suitable protease activities amount for example to2.25, 5 or 10 PE per ml of washing liquor. However, the proteaseactivity is not equal to zero.

An alternative test for establishing the proteolytic activity of theproteases is an optical measuring method, such as a photometric method.The appropriate test comprises the protease-dependent cleavage of thesubstrate protein casein. This is cleaved by the protease into amultitude of smaller partial products. The totality of these partialproducts has an increased absorption at 290 nm compared with uncleavedcasein, it being possible for this increased absorption to be determinedusing a photometer, and thus for a conclusion to be drawn regarding theenzymatic activity of the protease.

The protein concentration can be determined using known methods, forexample the BCA method (bicinchoninic acid;2,2′-bichinolyl-4,4′-dicarboxylic acid) or the Biuret method (A. G.Gornall, C. S. Bardawill and M. M. David, J. Biol. Chem., 177 (1948), p.751-766). The active protein concentration can be determined in thisregard by titrating the active centers using a suitable irreversibleinhibitor and determining the residual activity (cf. M. Bender et al.,J. Am. Chem. Soc. 88, 24 (1966), p. 5890-5913).

In addition to the amino acid alterations discussed above, proteases canhave other amino acid alterations, in particular amino acidsubstitutions, insertions or deletions. Such proteases are, for example,further developed by targeted genetic modification, i.e. by mutagenesismethods, and optimized for specific applications or with regard tospecific properties (for example with regard to their catalyticactivity, stability, etc.). Furthermore, nucleic acids can be introducedinto recombination approaches and can thus be used to generatecompletely novel proteases or other polypeptides.

The aim is to introduce targeted mutations such as substitutions,insertions or deletions into the known molecules in order, for example,to improve the cleaning performance of enzymes. For this purpose, inparticular the surface charges and/or the isoelectric point of themolecules and thus their interactions with the substrate can be altered.For instance, the net charge of the enzymes can be altered in order toinfluence the substrate binding, in particular for use in washing andcleaning agents. Alternatively or in addition, one or more correspondingmutations can increase the stability or catalytic activity of theprotease and thus improve its cleaning performance. Advantageousproperties of individual mutations, e.g. individual substitutions, cancomplement one another. A protease which has already been optimized withregard to specific properties, for example with respect to its stabilityduring storage, can therefore also be further developed.

For the description of substitutions relating to exactly one amino acidposition (amino acid exchanges), the following convention is usedherein: first, the naturally occurring amino acid is designated in theform of the internationally used one-letter code, followed by theassociated sequence position and finally the inserted amino acid.Several exchanges within the same polypeptide chain are separated byslashes. For insertions, additional amino acids are named following thesequence position. In the case of deletions, the missing amino acid isreplaced by a symbol, for example a star or a dash, or a 4 is indicatedbefore the corresponding position. For example, P9T describes thesubstitution of proline at position 9 by threonine, P9TH describes theinsertion of histidine following the amino acid threonine at position 9and P9* or ΔP9 describes the deletion of proline at position 9. Thisnomenclature is known to a person skilled in the field of enzymetechnology.

A protease may be characterized in that it is obtainable from a proteaseas described above as the starting molecule by one-time or multipleconservative amino acid substitution, the protease in the numberingaccording to SEQ ID NO:1 having the above-described amino acidsubstitutions. The term “conservative amino acid substitution” means theexchange (substitution) of one amino acid residue for another amino acidresidue, with this exchange not resulting in a change to the polarity orcharge at the position of the exchanged amino acid, e.g. the exchange ofa nonpolar amino acid residue for another nonpolar amino acid residue.Conservative amino acid substitutions include, for example: G=A=S,l=V=L=M, D=E, N=Q, K=R, Y=F, S=T, G=A=I=V=L=M=Y=F=W=P=S=T.

Alternatively or in addition, the protease is characterized in that itis obtainable from a protease as a starting molecule by fragmentation ordeletion, insertion or substitution mutagenesis and comprises an aminoacid sequence which matches the starting molecule over a length of atleast 200, 210, 220, 230, 240, 250, 260, 261, 262, 263, 264, 265, 266,267, 268, 269, 270, 271, 272, 273, or 274 or 275 contiguous amino acids,the amino acid substitution(s) which is/are described above and possiblycontained in the starting molecule, i.e. the substitutions at thepositions corresponding to positions 9, 29, 48, 101, 130, 131, 133, 144,216, 224, 252 and 271 in SEQ ID NO:1, and optionally the amino acidsubstitution 166M, 166Q or 166A at the position corresponding toposition 166, and/or at least one further amino acid substitution at atleast one of positions corresponding to positions 62, 133, 149, 156, 172or 217, still being present.

For instance, it is possible to delete individual amino acids at thetermini or in the loops of the enzyme without the proteolytic activitybeing lost or diminished in the process. Furthermore, such fragmentationor deletion, insertion or substitution mutagenesis can also, forexample, reduce the allergenicity of the enzymes concerned and thusimprove their overall applicability. Advantageously, the enzymes retaintheir proteolytic activity even after mutagenesis, i.e. theirproteolytic activity corresponds at least to that of the startingenzyme, i.e. in a non-limiting embodiment the proteolytic activity is atleast 80%, such as at least 90% of the activity of the starting enzyme.Other substitutions can also exhibit advantageous effects. Both singleand multiple contiguous amino acids can be exchanged for other aminoacids.

Alternatively or in addition, the protease is characterized in that itis obtainable from a protease as the starting molecule by one-time ormultiple conservative amino acid substitution, the protease having (i)the amino acid substitutions P9T, P9H, P9S or P9A and Q271E at thepositions corresponding to positions 9 and 271 according to SEQ ID NO:1,and (ii) at least one of the amino acid substitutions A29G, A48V, D101E,N130D, N130S, N130H, G131S, G131N, G131D, G131K, T133K, T133R, T133Y,N144K, N144L, N144A, S224A, S224T or N252S at the positionscorresponding to positions 29, 48, 101, 130, 131, 133, 144, 224 and 252according to SEQ ID NO:1, and optionally the amino acid substitution166M, 166Q or 166A at the position corresponding to position 166, and/orat least one further amino acid substitution at at least one of thepositions corresponding to positions 62, 133, 149, 156, 172 or 217.

In further embodiments, the protease is characterized in that it isobtainable from a protease as the starting molecule by fragmentation ordeletion, insertion or substitution mutagenesis and comprises an aminoacid sequence which matches the starting molecule over a length of atleast 200, 210, 220, 230, 240, 250, 260, 261, 262, 263, 264, 265, 266,267, 268, 269, 270, 271, 272, 273, or 274 or 275 contiguous amino acids,the protease comprising (i) the amino acid substitutions P9T, P9H, P9Sor P9A and Q271E at the positions corresponding to positions 9 and 271according to SEQ ID NO:1, and (ii) at least one of the amino acidsubstitutions A29G, A48V, D101E, N130D, N130S, N130H, G131S, G131N,G131D, G131K, T133K, T133R, T133Y, N144K, N144L, N144A, S224A, S224T orN252S at the positions corresponding to positions 29, 48, 101, 130, 131,133, 144, 224 and 252 according to SEQ ID NO:1, and optionally the aminoacid substitution 166M, 166Q or 166A at the position corresponding toposition 166, and/or at least one further amino acid substitution at atleast one of the positions corresponding to positions 62, 133, 149, 156,172 or 217.

The further amino acid positions are in this case defined by analignment of the amino acid sequence of a protease with the amino acidsequence of the protease from Bacillus pumilus, as given in SEQ ID NO:1.Furthermore, the assignment of the positions depends on the matureprotein. This assignment is in particular also to be used if the aminoacid sequence of a protease comprises a higher number of amino acidresidues than the protease from Bacillus pumilus according to SEQ IDNO:1. Proceeding from the above-mentioned positions in the amino acidsequence of the protease from Bacillus pumilus, the alteration positionsin a protease are those which are assigned to precisely these positionsin an alignment.

Advantageous positions for sequence alterations, in particularsubstitutions, of the protease from Bacillus pumilus, which are ofparticular significance when transferred to homologous positions of theproteases and which impart advantageous functional properties to theprotease are therefore the positions which correspond to positions 9,29, 48, 62, 101, 130, 131, 133, 144, 149, 156, 166, 172, 216, 217, 224,252 and 271 in SEQ ID NO:1 in an alignment, i.e. in the numberingaccording to SEQ ID NO:1. At the positions mentioned, the followingamino acid residues are present in the wild-type molecule of theprotease from Bacillus pumilus: P9, A29, A48, Q62, D101, N130, G131,T133, N144, V149, S156, G166, D172, S216, Y217, S224, N252 and Q271.

Further confirmation of the correct assignment of the amino acids to bealtered, i.e. in particular their functional correspondence, can beprovided by comparative experiments, according to which the twopositions assigned to one another on the basis of an alignment arealtered in the same way in both compared proteases, and observations aremade as to whether the enzymatic activity is altered in the same way inboth cases. If, for example, an amino acid exchange in a specificposition of the protease from Bacillus pumilus according to SEQ ID NO:1is accompanied by an alteration of an enzymatic parameter, for examplean increase in the K_(M) value, and a corresponding alteration of theenzymatic parameter, for example likewise an increase in the K_(M)value, is observed in a protease variant of which the amino acidexchange has been achieved by the same introduced amino acid, this cantherefore be considered to be confirmation of the correct assignment.

All of these aspects are also applicable to the method for producing aprotease. Accordingly, a method further comprises one or more of thefollowing method steps:

-   -   a) Introducing one-time or multiple conservative amino acid        substitution, the protease comprising    -   (i) the amino acid substitutions P9T, P9H, P9S or P9A and Q271E        at the positions corresponding to positions 9 and 271 according        to SEQ ID NO:1, and    -   (ii) at least one of the amino acid substitutions A29G, A48V,        D101E, N130D, N130S, N130H, G131S, G131N, G131D, G131K, T133K,        T133R, T133Y, N144K, N144L, N144A, S224A, S224T or N252S at the        positions corresponding to positions 29, 48, 101, 130, 131, 133,        144, 224 and 252 according to SEQ ID NO:1;    -   b) Altering the amino acid sequence by fragmentation or        deletion, insertion or substitution mutagenesis such that the        protease comprises an amino acid sequence which matches the        starting molecule over a length of at least 200, 210, 220, 230,        240, 250, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270,        271, 272, 273, or 274 or 275 contiguous amino acids, the        protease comprising    -   (i) the amino acid substitutions P9T, P9H, P9S or P9A and Q271E        at the positions corresponding to positions 9 and 271 according        to SEQ ID NO:1, and    -   (ii) at least one of the amino acid substitutions A29G, A48V,        D101E, N130D, N130S, N130H, G131S, G131N, G131D, G131K, T133K,        T133R, T133Y, N144K, N144L, N144A, S224A, S224T or N252S at the        positions corresponding to positions 29, 48, 101, 130, 131, 133,        144, 224 and 252 according to SEQ ID NO:1.

In various embodiments of this method, this comprises one or more of thefollowing steps:

-   -   a) Introducing one-time or multiple conservative amino acid        substitution into the protease, the protease comprising the        substitutions 9T, 130D, 144K, 252T and 271E at the positions        corresponding to positions 9, 130, 144, 252 and 271; and        optionally the amino acid substitution 166M, 166Q or 166A at the        position corresponding to position 166, and/or at least one        further amino acid substitution at at least one of the positions        corresponding to positions 62, 133, 149, 156, 172 or 217;    -   b) Altering the amino acid sequence by fragmentation or        deletion, insertion or substitution mutagenesis such that the        protease comprises an amino acid sequence which matches the        starting molecule over a length of at least 200, 210, 220, 230,        240, 250, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270,        271, 272, 273 or 274 contiguous amino acids, the protease        comprising the substitutions 9T, 130D, 144K, 252T and 271E at        the positions corresponding to positions 9, 130, 144, 252 and        271; and optionally the amino acid substitution 166M, 166Q or        166A at the position corresponding to position 166, and/or at        least one further amino acid substitution at at least one of the        positions corresponding to positions 62, 133, 149, 156, 172 or        217.

All embodiments also apply to the method.

In further embodiments, the protease or the protease produced by meansof a method is still at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%,91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%,97%, 97.5%, 98%, 98.5%, or 98.8% identical to the amino acid sequencegiven in SEQ ID NO:1 over its entire length. Alternatively, the proteaseor the protease produced by means of a method is still at least 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%,94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, or 98% identical to oneof the amino acid sequences given in SEQ ID Nos: 3-17 or 19-23 over itsentire length. The protease or the protease produced by means of amethod has an amino acid substitution at positions 9 and 271 and atleast one of the positions corresponding to positions 29, 48, 101, 130,131, 133, 144, 224 or 252, in each case based on the numbering accordingto SEQ ID NO:1. In more embodiments, the amino acid substitution is atleast one selected from the group consisting of P9T, P9H, P9S, P9A,A29G, A48V, D101E, N130D, N130S, N130H, G131S, G131N, G131D, G131K,T133K, T133R, T133Y, N144K, N144L, N144A, S224A, S224T, N252S and Q271E,in each case based on the numbering according to SEQ ID NO:1. In furtherembodiments, the protease further comprises a substitution at theposition 216, in particular S216C; in various embodiments it may beoptional for this position to not be substituted. The following aminoacid substitution variants are most particularly suitable: P9T, P9H, P9Sor P9A, in particular P9T, and Q271E and optionally also S216C combinedwith one of (i) N130D, G131S, T133Y and S224A; (ii) N130S, G131S, T133Y,N144L and S224A; (iii) N130D, G131N, T133K and N144K; (iv) N130H, G131N,N144K and S224A; (v) A29G, N130D, G131N and T133R; (vi) A29G, D101E,N130S, G131S, S224T and N252S; (vii) N130H, G131S and S224A; (viii)N130D, G131S, T133K and S224A; (ix) A29G, D101E, N130D, G131K and S224A;(x) N130D, G131N T133K, N144L and N252S; (xi) G131S, N144K and S224T;(xii) D101E, N130D, G131S, T133Y, N144A and S224A; (xiii) A29G, D101E,N130S, S224T and N252S; (xiv) A48V, G131S, T133R and S224A; or (xv)G131D, T133R and S224A, the numbering being based in each case on thenumbering according to SEQ ID NO:1.

In further embodiments, the protease or the protease produced by meansof a method has the amino acid substitutions 9T, 130D, 144K, 252T and271E at the positions corresponding to positions 9, 130, 144, 252 and271; and optionally the amino acid substitution 166M, 166Q or 166A atthe position corresponding to position 166, and/or at least one furtheramino acid substitution at at least one of the positions correspondingto positions 62, 133, 149, 156, 172 or 217, in each case based on thenumbering according to SEQ ID NO:1. Examples thereof are the followingamino acid substitution variants: P9T and Q271E combined with one of (i)N130D, N144K, N252T and G166M; (ii) N130D, N144K, N252T and G166Q; (iii)N130D, N144K, N252T and G166A; (iv) N130D, N144K, N252T and S156G; or(v) N130D, N144K, N252T and Y217M, the numbering being based in eachcase on the numbering according to SEQ ID NO:1 and the variantsdescribed in the examples.

A protease described above that may be additionally stabilized, inparticular by one or more mutations, for example substitutions, or bycoupling to a polymer. An increase in stability during storage and/orduring use, for example in the washing process, leads to longerenzymatic activity and thus improves the cleaning performance. Inprinciple, all stabilization options which are described in the priorart and/or are appropriate are considered. Those stabilizations aresuitable which are achieved by mutations of the enzyme itself, sincesuch stabilizations do not require any further work steps following therecovery of the enzyme. Examples of sequence alterations suitable forthis purpose are mentioned above. Further suitable sequence alterationsare known from the prior art.

Further possibilities for stabilization are, for example:

-   -   Altering the binding of metal ions, in particular the calcium        binding sites, for example by exchanging one or more of the        amino acids involved in the calcium binding with one or more        negatively charged amino acids and/or by introducing sequence        alterations into at least one of the results of the two amino        acids arginine/glycine;    -   Protecting against the influence of denaturing agents such as        surfactants by mutations that cause an alteration of the amino        acid sequence on or at the surface of the protein;    -   Replacing amino acids which are close to the N-terminus with        those likely to contact the rest of the molecule via        non-covalent interactions, thus contributing to the maintenance        of the globular structure.

Non-limiting embodiments are those in which the enzyme is stabilized inseveral ways, since several stabilizing mutations have an additive orsynergistic effect.

A protease as described above that may be characterized in that it hasat least one chemical modification. A protease having such an alterationis called a derivative, i.e. the protease is derivatized.

In the context of the present application, derivatives are thusunderstood to mean those proteins of which the pure amino acid chain hasbeen chemically modified. Such derivatizations can be achieved, forexample, in vivo by the host cell that expresses the protein. In thisregard, couplings of low-molecular-weight compounds such as lipids oroligosaccharides are particularly noteworthy. However, thederivatizations may also be carried out in vitro, for example by thechemical conversion of a side chain of an amino acid or by covalentbonding of another compound to the protein. For example, it is possibleto couple amines to carboxyl groups of an enzyme in order to alter theisoelectric point. Another such compound may also be another proteinthat is bound to a protein via bifunctional chemical compounds, forexample. Derivatization is also understood to mean the covalent bondingto a macromolecular carrier or a non-covalent inclusion in suitablemacromolecular cage structures. Derivatizations may, for example, affectthe substrate specificity or bonding strength to the substrate or causea temporary blockage of the enzymatic activity when the coupledsubstance is an inhibitor. This can be expedient, for example, for theperiod of the storage. Such modifications may further affect thestability or enzymatic activity. They can also be used to reduce theallergenicity and/or immunogenicity of the protein and thus, forexample, increase its skin compatibility. For example, couplings withmacromolecular compounds, for example polyethylene glycol, can improvethe protein in terms of stability and/or skin compatibility.

Derivatives of a protein can also be understood in the broadest sense tomean preparations of these proteins. Depending on the recovery,processing or preparation, a protein can be combined with various othersubstances, for example from the culture of the producingmicroorganisms. A protein may also have been deliberately added to othersubstances, for example to increase its storage stability. This is alsoirrespective of whether or not it actually exhibits this enzymaticactivity in a particular preparation. This is because it may be desiredthat it has no or only low activity during storage, and exhibits itsenzymatic function only at the time of use. This can be controlled viaappropriate accompanying substances, for example. In particular, thejoint preparation of proteases with specific inhibitors is possible inthis regard.

Of all the proteases or protease variants and/or derivatives describedabove, those of which the storage stability corresponds to at least oneof those of the proteases according to SEQ ID Nos: 3-17 or 19-23, and/orof which the cleaning performance corresponds to at least one of thoseof the proteases according to SEQ ID Nos: 3-17 or 19-23, areparticularly possible, the cleaning performance being determined in awashing system as described above.

A nucleic acid which codes for a protease, as well as to a vectorcontaining such a nucleic acid, in particular a cloning vector or anexpression vector.

These may be DNA or RNA molecules. They can be present as a singlestrand, as a single strand that is complementary to this single strand,or as a double strand. In particular in the case of DNA molecules, thesequences of the two complementary strands must be taken into account inall three possible reading frames. Furthermore, it should be noted thatdifferent codons, i.e. base triplets, can code for the same amino acidssuch that a particular amino acid sequence can be coded by a pluralityof different nucleic acids. Due to this degeneracy of the genetic code,all of the nucleic acid sequences which can code any of the proteasesdescribed above are included. A person skilled in the art is able todetermine these nucleic acid sequences unequivocally since, despite thedegeneracy of the genetic code, defined amino acids can be assigned toindividual codons. Therefore, a person skilled in the art proceedingfrom an amino acid sequence can easily determine nucleic acids codingfor said amino acid sequence. Furthermore, in the case of nucleic acids,one or more codons may be replaced by synonymous codons. This aspectrelates in particular to the heterologous expression of the enzymes. Forinstance, every organism, for example a host cell of a productionstrain, has a particular codon usage. Codon usage is understood to meanthe translation of the genetic code into amino acids by the particularorganism. Bottlenecks can occur in the protein biosynthesis if thecodons on the nucleic acid in the organism are faced with acomparatively small number of loaded tRNA molecules. Although coding forthe same amino acid, this results in a codon being translated lessefficiently in the organism than a synonymous codon coding for the sameamino acid. Due to the presence of a higher number of tRNA molecules forthe synonymous codon, it can be translated more efficiently in theorganism.

Using methods which are currently generally known, such as chemicalsynthesis or the polymerase chain reaction (PCR), in conjunction withmolecular-biological and/or protein-chemical standard methods, it ispossible for a person skilled in the art to produce the correspondingnucleic acids and even complete genes on the basis of known DNA and/oramino acid sequences. Such methods are known for example from Sambrook,J., Fritsch, E. F. and Maniatis, T. 2001. Molecular cloning: alaboratory manual, 3. Edition Cold Spring Laboratory Press.

Within the meaning herein, vectors are understood to mean elementsconsisting of nucleic acids, which elements contain a nucleic acid asthe characteristic nucleic acid region. They are able to establish theseas a stable genetic element in a species or cell line over severalgenerations or cell divisions. Vectors are special plasmids, i.e.circular genetic elements, in particular when used in bacteria. Anucleic acid is cloned into a vector. The vectors include, for example,those originating from bacterial plasmids, viruses or bacteriophages, orpredominantly synthetic vectors or plasmids with elements of a widevariety of origins. With the additional genetic elements present in eachcase, vectors are able to establish themselves as stable units in thecorresponding host cells over several generations. They may be presentas separate units in an extrachromosomal manner or integrated into achromosome or chromosomal DNA.

Expression vectors comprise nucleic acid sequences which enable them toreplicate in the host cells containing them, such as microorganisms,e.g. bacteria, and to express a contained nucleic acid there. Theexpression is in particular influenced by the promoter(s) that regulatethe transcription. In principle, the expression can take place by thenatural promoter originally located before the nucleic acid to beexpressed, but also by a promoter of the host cell provided on theexpression vector or also by a modified or completely different promoterof another organism or another host cell. In the present case, at leastone promoter is provided for the expression of a nucleic acid and usedfor the expression thereof. Furthermore, expression vectors can beregulatable, for example by changing the cultivation conditions or whena specific cell density of the host cells containing them is reached orby addition of specific substances, in particular activators of geneexpression. An example of such a substance is the galactose derivativeisopropyl-β-D-thiogalactopyranoside (IPTG), which is used as anactivator of the bacterial lactose operon (lac operon). In contrast withexpression vectors, the nucleic acid contained is not expressed incloning vectors.

A non-human host cell which contains a nucleic acid or a vector or whichcontains a protease, in particular one which secretes the protease intothe medium surrounding the host cell. In a non-limiting embodiment, anucleic acid or a vector is transformed into a microorganism, which thenrepresents a host cell. Alternatively, individual components, i.e.nucleic acid parts or fragments of a nucleic acid can be introduced intoa host cell such that the resulting host cell contains a nucleic acid ora vector. This procedure is particularly suitable when the host cellalready contains one or more constituents of a nucleic acid or a vectorand the further constituents are then supplemented accordingly. Methodsfor transforming cells are established in the prior art and are wellknown to a person skilled in the art. In principle, all cells, i.e.prokaryotic or eukaryotic cells, are suitable as host cells. Host cellsthat can be managed in a genetically advantageous manner, for example interms of the transformation with the nucleic acid or the vector and thestable establishment thereof, are possible, for example unicellularfungi or bacteria. Furthermore, host cells are characterized by goodmicrobiological and biotechnological manageability. This relates, forexample, to easy cultivation, high growth rates, low requirements forfermentation media and good production and secretion rates for foreignproteins. Non-limiting host cells secrete the (transgenically) expressedprotein into the medium surrounding the host cells. Furthermore, theproteases can be modified by the cells producing them after theirproduction, for example by attachment of sugar molecules, formylations,aminations, etc. Such post-translational modifications can functionallyinfluence the protease.

Other embodiments are those host cells of which the activity can beregulated on account of genetic regulatory elements, which are, forexample, made available on the vector but may also be present in thesecells from the outset. These host cells may be induced to express forexample by the controlled addition of chemical compounds which are usedas activators, by changing the cultivation conditions, or upon reachinga specific cell density. This enables an economical production of theproteins. An example of such a compound is IPTG as described above.

Prokaryotic or bacterial cells are suitable host cells. Bacteria arecharacterized by short generation times and low demands on cultivationconditions. As a result, cost-effective cultivation methods orproduction methods can be established. In addition, a person skilled inthe art has a wealth of experience in the case of bacteria infermentation technology. For a specific production, gram-negative orgram-positive bacteria may be suitable for a wide variety of reasons tobe determined experimentally in individual cases, such as nutrientsources, product formation rate, time requirement, etc.

In the case of gram-negative bacteria, such as Escherichia coli, a largenumber of proteins are secreted into the periplasmic space, i.e. intothe compartment between the two membranes enclosing the cells. This maybe advantageous for particular applications. Furthermore, gram-negativebacteria can also be designed such that they eject the expressedproteins not only into the periplasmic space, but into the mediumsurrounding the bacterium. In contrast, gram-positive bacteria such asbacilli or actinomycetes or other representatives of Actinomycetaleshave no outer membrane, and therefore secreted proteins are releasedimmediately into the medium surrounding the bacteria, usually thenutrient medium, from which the expressed proteins can be purified. Theycan be isolated directly from the medium or further processed. Inaddition, gram-positive bacteria are related or identical to most of theorigin organisms for technically significant enzymes and usually evenform comparable enzymes, meaning that they have a similar codon usageand the protein synthesis apparatus is naturally aligned accordingly.

Host cells may be altered in terms of their requirements for the cultureconditions, may have different or additional selection markers or mayexpress other or additional proteins. In particular, this may alsoinvolve those host cells which transgenically express several proteinsor enzymes.

Microorganisms, in particular fermentable microorganisms, such as thoseof the genus Bacillus, and leads to it being possible to produceproteins by the use of such microorganisms. Such microorganisms thenrepresent host cells.

In a further embodiment, the host cell is characterized in that it is abacterium, such as one selected from the group of the genera ofEscherichia, Klebsiella, Bacillus, Staphylococcus, Corynebacterium,Arthrobacter, Streptomyces, Stenotrophomonas and Pseudomonas, moresuitably one selected from the group of Escherichia coli, Klebsiellaplanticola, Bacillus licheniformis, Bacillus lentus, Bacillusamyloliquefaciens, Bacillus subtilis, Bacillus alcalophilus, Bacillusglobigii, Bacillus gibsonii, Bacillus clausii, Bacillus halodurans,Bacillus pumilus, Staphylococcus carnosus, Corynebacterium glutamicum,Arthrobacter oxidans, Streptomyces lividans, Streptomyces coelicolor andStenotrophomonas maltophilia.

The host cell may also be a eukaryotic cell, however, which ischaracterized in that it has a cell nucleus. A host cell may becharacterized in that it has a cell nucleus. In contrast withprokaryotic cells, eukaryotic cells are capable of post-translationallymodifying the protein formed. Examples thereof are fungi such asactinomycetes or yeasts such as Saccharomyces or Kluyveromyces. This canbe particularly advantageous, for example, if the proteins are toundergo specific modifications in connection with their synthesis thatmake such systems possible. Modifications carried out by eukaryoticsystems, in particular in connection with the protein synthesis,include, for example, the binding of low-molecular-weight compounds suchas membrane anchors or oligosaccharides. Such oligosaccharidemodifications may be desirable, for example, to lower the allergenicityof an expressed protein. Co-expression with the enzymes naturally formedby such cells, such as cellulases, may be advantageous. Furthermore, forexample, thermophilic fungal expression systems may be particularlysuitable for the expression of temperature-resistant proteins orvariants.

The host cells are cultivated and fermented in the conventional way, forexample in discontinuous or continuous systems. In the first case, asuitable nutrient medium is inoculated with the host cells and theproduct is harvested from the medium after a period to be determinedexperimentally. Continuous fermentations are characterized by theachievement of a flow equilibrium, in which cells partially die over acomparatively long period of time but also grow back and the proteinformed can be removed from the medium at the same time.

Host cells are used to produce proteases. A method for preparing aprotease, may include

a) cultivating a host cell, and

b) isolating the protease from the culture medium or from the host cell.

This subject matter comprises fermentation processes. Fermentationprocesses are known per se from the prior art and represent the actuallarge-scale production step, usually followed by a suitable purificationmethod of the produced product, for example the proteases. Allfermentation processes which are based on a corresponding method forproducing a protease constitute embodiments of this subject matter.

Fermentation processes which are characterized in that the fermentationis carried out via a feed strategy shall be considered in particular. Inthis case, the media components that are consumed by the continuouscultivation are added. As a result, considerable increases can beachieved both in the cell density and in the cell mass or dry massand/or in particular in the activity of the protease of interest.Furthermore, the fermentation can also be designed in such a way thatundesired metabolic products are filtered out or neutralized by addingbuffers or suitable counter ions.

The protease produced can be harvested from the fermentation medium.Such a fermentation process is better than isolation of the proteasefrom the host cell, i.e. product preparation from the cell mass (drymatter), but requires the provision of suitable host cells or one ormore suitable secretion markers or mechanisms and/or transport systemsfor the host cells to secrete the protease into the fermentation medium.Without secretion, the protease can alternatively be isolated from thehost cell, i.e. purified from the cell mass, for example byprecipitation with ammonium sulphate or ethanol, or by chromatographicpurification.

All of the above-mentioned aspects can be combined into methods in orderto produce protease.

An agent may be characterized in that it contains a protease asdescribed above. The agent is a washing or cleaning agent.

This subject matter covers all conceivable types of washing or cleaningagents, both concentrates and agents to be used undiluted, for use on acommercial scale, in washing machines or for hand washing or cleaning.These include, for example, washing agents for textiles, carpets, ornatural fibers, for which the term washing agent is used. These alsoinclude, for example, dishwashing detergents for dishwashers or manualdishwashing detergents or cleaners for hard surfaces such as metal,glass, porcelain, ceramics, tiles, stone, painted surfaces, plastics,wood or leather, for which the term cleaning agent is used, i.e. inaddition to manual and mechanical dishwashing detergents, also, forexample, scouring agents, glass cleaners, toilet rim blocks, etc. Thewashing and cleaning agents also include auxiliary washing agents whichare added to the actual washing agent during manual or automatic textilewashing in order to achieve a further effect. Furthermore, washing andcleaning agents also include textile pre-treatment and post-treatmentagents, i.e. those agents with which the item of laundry is brought intocontact before the actual washing cycle, for example to loosen stubbornsoiling, and also those agents which give the laundry further desirableproperties such as a pleasant feel, crease resistance or low staticcharge in a step subsequent to the actual textile wash. Inter alia,softeners are included in the last-mentioned agents.

The washing or cleaning agents, which may be in the form of powderedsolids, in further-compacted particulate form, homogeneous solutions orsuspensions, may contain, in addition to a protease, all knowningredients conventional in such agents, with at least one otheringredient being present in the agent. The agents may in particularcontain surfactants, builders, peroxygen compounds or bleach activators.They may also contain water-miscible organic solvents, further enzymes,sequestering agents, electrolytes, pH regulators and/or furtherauxiliaries such as optical brighteners, graying inhibitors, foamregulators, as well as dyes and fragrances, and combinations thereof.

In particular, a combination of a protease with one or more furtheringredients of the agent is advantageous, since, in embodiments, such anagent has improved cleaning performance by virtue of resultingsynergisms. In particular, combining a protease with a surfactant and/ora builder and/or a peroxygen compound and/or a bleach activator canresult in such a synergism. However, in embodiments, the agent may notcontain boric acid.

Advantageous ingredients of agents are disclosed in international patentapplication WO2009/121725, starting at the penultimate paragraph of page5 and ending after the second paragraph on page 13. Reference isexpressly made to this disclosure and the disclosure therein isincorporated in the present patent application by reference.

An agent advantageously contains the protease in an amount of from 2 μgto 20 mg, such as from 5 μg to 17.5 mg, alternatively from 20 μg to 15mg or from 50 μg to 10 mg per g of the agent. In various embodiments,the concentration of the protease (active enzyme) described herein inthe agent is >0 to 1 wt. %, such as 0.001 to 0.1 wt. %, based on thetotal weight of the agent or composition. Further, the proteasecontained in the agent, and/or other ingredients of the agent, may becoated with a substance which is impermeable to the enzyme at roomtemperature or in the absence of water, and which becomes permeable tothe enzyme under conditions of use of the agent. Such an embodiment isthus characterized in that the protease is coated with a substance whichis impermeable to the protease at room temperature or in the absence ofwater. Furthermore, the washing or cleaning agent itself can be packedin a container, such as an air-permeable container, from which it isreleased shortly before use or during the washing process.

In further embodiments, the agent is characterized in that it

-   -   (a) is present in solid form, in particular as a flowable powder        having a bulk density of from 300 g/l to 1200 g/l, in particular        from 500 g/l to 900 g/l, or    -   (b) is present in paste or liquid form, and/or    -   (c) is present in the form of a gel or in the form of dosing        pouches, and/or    -   (d) is present as a single-component system, or    -   (e) is divided into a plurality of components.

These embodiments include all solid, powdered, liquid, gel or pasteadministration forms of agents, which may optionally also consist of aplurality of phases and can be present in compressed or uncompressedform. The agent may be present as a flowable powder, in particularhaving a bulk density of from 300 g/l to 1200 g/l, in particular from500 g/l to 900 g/l or from 600 g/l to 850 g/l. The solid administrationforms of the agent also include extrudates, granules, tablets orpouches. Alternatively, the agent may also be in liquid, gel or pasteform, for example in the form of a non-aqueous liquid washing agent or anon-aqueous paste or in the form of an aqueous liquid washing agent or awater-containing paste. The agent may also be present as a one-componentsystem. Such agents consist of one phase. Alternatively, an agent mayalso consist of a plurality of phases. Such an agent is thereforedivided into a plurality of components.

Washing or cleaning agents may contain only one protease.

Alternatively, they may also contain other hydrolytic enzymes or otherenzymes in a concentration that is expedient for the effectiveness ofthe agent. A further embodiment is therefore represented by agents whichfurther comprise one or more further enzymes. Further enzymes which canbe used are all enzymes which can exhibit catalytic activity in theagent, in particular a lipase, amylase, cellulase, hemicellulase,mannanase, tannase, xylanase, xanthanase, xytoglucanase, β-glucosidase,pectinase, carrageenase, perhydrolase, oxidase, oxidoreductase oranother protease, which is different from the proteases, as well asmixtures thereof. Further enzymes are advantageously contained in theagent in an amount of from 1×10⁻⁸ to 5 wt. % based on active protein.Each further enzyme may be contained in agents in an amount of from1×10⁻⁷ to 3 wt. %, from 0.00001 to 1 wt. %, from 0.00005 to 0.5 wt. %,from 0.0001 to 0.1 wt. % and such as from 0.0001 to 0.05 wt. %, based onactive protein. In a non-limiting embodiment, the enzymes exhibitsynergistic cleaning performance against specific stains or spots, i.e.the enzymes contained in the agent composition support one another intheir cleaning performance. There is such synergism between the proteaseand a further enzyme of an agent, including in particular between saidprotease and an amylase and/or a lipase and/or a mannanase and/or acellulase and/or a pectinase. Synergistic effects can arise not onlybetween different enzymes, but also between one or more enzymes andother ingredients of the agent.

In the cleaning agents described herein, the enzymes to be used mayfurthermore be formulated together with accompanying substances, forexample from fermentation. In liquid formulations, the enzymes are usedas enzyme liquid formulations.

The enzymes are generally not provided in the form of pure protein, butrather in the form of stabilized, storable and transportablepreparations. These pre-formulated preparations include, for example,the solid preparations obtained through granulation, extrusion, orlyophilization or, in particular in the case of liquid or gel agents,solutions of the enzymes, advantageously maximally concentrated,low-water, and/or supplemented with stabilizers or other auxiliaries.

Alternatively, the enzymes can also be encapsulated, for both the solidand the liquid administration form, for example by spray-drying orextrusion of the enzyme solution together with a natural polymer or inthe form of capsules, for example those in which the enzymes areenclosed in a set gel, or in those of the core-shell type, in which anenzyme-containing core is coated with a water-, air-, and/orchemical-impermeable protective layer. Other active ingredients such asstabilizers, emulsifiers, pigments, bleaching agents, or dyes canadditionally be applied in overlaid layers. Such capsules are appliedusing inherently known methods, for example by shaking or rollgranulation or in fluidized bed processes. Such granules areadvantageously low in dust, for example due to the application ofpolymeric film-formers, and are stable in storage due to the coating.

Moreover, it is possible to formulate two or more enzymes together, suchthat a single granule exhibits a plurality of enzyme activities.

The enzymes can also be incorporated in water-soluble films, such asthose used in the formulation of washing and cleaning agents in a unitdosage form. Such a film allows the release of the enzymes followingcontact with water. As used herein, “water-soluble” refers to a filmstructure that is completely water-soluble. In a non-limitingembodiment, such a film consists of (fully or partially hydrolyzed)polyvinyl alcohol (PVA).

A method for cleaning textiles or hard surfaces may be characterized inthat an agent is used in at least one method step, or in that a proteasebecomes catalytically active in at least one method step, in particularsuch that the protease is used in an amount of from 40 μg to 4 g, suchas from 50 μg to 3 g, alternatively from 100 μg to 2 g, or from 200 μgto 1 g.

In various embodiments, the method described above is characterized inthat the protease is used at a temperature of from 0 to 100° C., such as0 to 60° C., alternatively 20 to 40° C. or at 20 or 25° C.

These include both manual and mechanical methods. Methods for cleaningtextiles are generally characterized by the fact that, in a plurality ofmethod steps, various cleaning-active substances are applied to thematerial to be cleaned and washed off after the exposure time, or inthat the material to be cleaned is otherwise treated with a washingagent or a solution or dilution of this agent. The same applies tomethods for cleaning all materials other than textiles, in particularhard surfaces. All conceivable washing or cleaning methods can beenhanced in at least one of the method steps by the use of a washing orcleaning agent or a protease, and then represent embodiments. Allaspects, objects, and embodiments described for the protease and agentscontaining it are also applicable to this subject matter. Therefore,reference is expressly made at this point to the disclosure at theappropriate point with the note that this disclosure also applies to theabove-described method.

Since proteases naturally already have hydrolytic activity and alsoexhibit this in media which otherwise have no cleaning power, forexample in a simple buffer, a single and/or the sole step of such amethod can consist in the protease, which is the only cleaning-activecomponent, being brought into contact with the stain, in a buffersolution or in water. This represents a further embodiment of thissubject matter.

Alternative embodiments are also represented by methods for treatingtextile raw materials or for textile care, in which a protease becomesactive in at least one method step. Among these, methods for textile rawmaterials, fibers or textiles with natural components are suitable, andespecially for those with wool or silk.

Finally, the proteases described herein may be used in washing orcleaning agents, for example as described above, for the (improved)removal of protein-containing stains, for example from textiles or hardsurfaces. In embodiments of this use, the protease in the washing orcleaning agent is stored for 3 or more days, 4 or more days, 7 or moredays, 10 or more days, 12 or more days, 14 or more days, 21 or more daysor 28 or more days before a washing or cleaning process.

All aspects, objects, and embodiments described for the protease andagents containing it are also applicable to this subject matter.Therefore, reference is expressly made at this point to the disclosureat the appropriate point with the note that this disclosure also appliesto the above-described use.

Examples

Overview of the Mutations:

A subtilisin-type alkaline protease from Bacillus pumilus may be useableherein. From this protease (SEQ ID NO:1), variants were produced byrandom mutagenesis, which were then screened, inter alia for improvedwashing performance and/or enzyme stability. In this way, twoperformance-enhanced mutants (mutant 1 [SEQ ID NO:18] and mutant 2 [SEQID NO:2]) were generated from the wild-type protease mentioned above ina first round by error-prone mutagenesis. Both of these mutants weresubject to an independent, second error-prone round. In this secondround of mutation, mutants 3-17 according to SEQ ID Nos. 3-17 weregenerated. Therefore, all of mutants 3-17 mentioned here also carry atleast some of the mutations of mutants 1 or 2. In a third round, anotherperformance-enhanced mutant (mutant 18 [SEQ ID NO:19]) was generated byerror-prone mutagenesis. This mutant was subject to a fourth error-proneround. In this fourth round of mutation, mutants 19-23 according to SEQID Nos. 20-24 were generated. Therefore, all of mutants 19-23 mentionedhere also carry the mutations of mutant 18.

SEQ ID Variant Amino acid substitutions relative to SEQ ID NO: 1 NO.Mutant 1 P9T Q271E S216C 18 Mutant 2 P9T Q271E S216C T133R S224A 2Mutant 3 P9T Q271E S216C N130D G131S T133Y S224A 3 Mutant 4 P9T Q271ES216C N130S G131S T133Y N144L S224A 4 Mutant 5 P9T Q271E S216C N130DG131N T133K N144k 5 Mutant 6 P9T Q271E S216C N130H G131N N144K S224A 6Mutant 7 P9T Q271E S216C A29G N130D G131N T133R 7 Mutant 8 P9T Q271ES216C A29G D101E N130S G131S S224T N252S 8 Mutant 9 P9T Q271E S216CN130H G131S S224A 9 Mutant P9T Q271E S216C N130D G131S T133K S224A 10Mutant P9T Q271E S216C A29G D101E N130D G131K S224A 11 Mutant P9T Q271ES216C N130D G131N T133K N144L N252S 12 Mutant P9T Q271E S216C G131SN144K S224T 13 Mutant P9T Q271E S216C D101E N130D G131S T133Y N144AS224A 14 Mutant P9T Q271E S216C A29G D101E N130S S224T N252S 15 MutantP9T Q271E S216C A48V G131S T133R S224A 16 Mutant P9T Q271E S216C G131DT133R S224A 17 Mutant P9T Q271E N130D N144K N252T 19 Mutant P9T Q271EN130D N144K N252T G166M 20 Mutant P9T Q271E N130D N144K N252T G166Q 21Mutant P9T Q271E N130D N144K N252T G166A 22 Mutant P9T Q271E N130D N144KN252T Y217M 23 Mutant P9T Q271E N130D N144K N252T S156G 24 Mutant P9TQ271E N130D N144K N252T D172P Mutant P9T Q271E N130D N144K N252T V149IMutant P9T Q271E N130D N144K N252T Q62S Mutant P9T Q271E N130D N144KN252T Y217M T133R Mutant P9T Q271E N144K N252T Y217M Mutant P9T Q271EN130D N144K N252T S156QWashing Agent Matrix Used

The following washing agent matrices (commercially available, withoutenzymes, opt. brighteners, perfume and dyes) were used for the washingtest:

Storage Stability Test 1:

Wt. % of active Wt. % of active Chemical name substance in the rawsubstance in the Dem ineralized water 100 Remainder Alkyl benzenesulfonic acid 96 4.4 Anionic surfactants 70 5.6 C12-C18 fatty acid Nasalt 30 2.4 Non-ionic surfactants 100 4.4 Phosphonates 40 0.2 Citricacid 100 1.4 NaOH 50 0.95 Defoamer t.q. 0.01 Glycerol 100 2 Preservative100 0.08 Ethanol 93 1 Without opt. brighteners, perfume, dye andenzymes. Dosage 4.7 g/LStorage Stability Test 2:

Wt. % of active Wt. % of active Chemical name substance in the rawsubstance in the Demineralized water 100 Remainder Alkyl benzenesulfonic acid 96 12-18 Anionic surfactants 70 4-8 C12-C18 fatty acid Nasalt 30 2-4 Non-ionic surfactants 100  8-14 Phosphonate 60 0.5-2  Citric acid 100 3-5 NaOH 50 0.5-2   Defoamer 100 <1% Glycerol 99.5 1-31,2-propanediol 100  8-12 Monoethanolamine 100 4-8 Soil repellentpolymer 30 0.5-1   Without opt. brighteners, perfume, dye and enzymes.Dosage 3.17 g/LProtease Activity Assays

The activity of the protease is determined by the release of thechromophore para-nitroaniline from the substrate succinylalanine-alanine-proline-phenylalanine-para-nitroanilide (AAPFpNA; BachemL-1400). The release of the pNA causes an increase in absorbance at 410nm, the temporal progression of which is a measure of the enzymaticactivity.

The measurement was carried out at a temperature of 25° C., a pH of 8.6,and a wavelength of 410 nm. The measuring time was 5 minutes with ameasuring interval of from 20 to 60 seconds.

Measurement approach:

10 μL AAPF solution (70 mg/mL)

1000 μL Tris/HCl (0.1 M, pH 8.6 with 0.1% Brij 35)

10 μL diluted protease solution

Kinetics created over 5 min at 25° C. (410 nm)

Storage Stability Test and Results

The proteases were stirred into a washing agent matrix at the same levelof activity and stored at 30° C. By means of a conventional activityassay for proteases (hydrolysis of suc-AAPF-pNA), the starting activityand the residual activity of the protease are measured after 4 weeks'storage at 30° C. In order to generate harsh conditions, the proteaseswere stored in a washing agent matrix without a stabilizer (boric acid).

The proteases were generated in shake flask supernatants from Bacillussubtilis. They were diluted to an equal level of activity. 50% washingagent matrix without boric acid was added to 50% of appropriatelydiluted Bacillus subtilis protease supernatant and mixed well. Thesealed glasses were incubated at 30° C. At the time of sampling, apredetermined amount of matrix/protease mixture was removed anddissolved by stirring for 20 min at RT in the sample buffer (0.1 MTris/HCl, pH 8.6). The AAPF assay is then carried out as describedabove.

In the first storage stability test, 15 mutants had been found to beadvantageous. The activity is shown in % of the residual activity of thestarting variants (mutant 1 according to SEQ ID NO:18 or mutant 2according to SEQ ID NO:2) after 4 weeks' storage at 30° C. Mutants 3-15relate to starting mutant 1 (SEQ ID NO:18), and mutants 16 and 17 relateto starting mutant 2 (SEQ ID NO:2).

Variant SEQ ID NO: Residual activity Mutant 1 18 100% Mutant 3 3 153%Mutant 4 4 148% Mutant 5 5 150% Mutant 6 6 133% Mutant 7 7 154% Mutant 88 132% Mutant 9 9 147% Mutant 10 10 146% Mutant 11 11 132% Mutant 12 12144% Mutant 13 13 136% Mutant 14 14 138% Mutant 15 15 132% Mutant 2 2100% Mutant 16 16 121% Mutant 17 17 110%

It can be seen that all the mutants exhibit greatly improved stabilitywithout the addition of boric acid in comparison with the respectivestarting mutants according to SEQ ID Nos. 18 and 2. All the mutantsexhibit a washing performance that is comparable to the wild typeaccording to SEQ ID NO:1, i.e. they are at most 10% worse in terms ofwashing performance, which is within the measurement fluctuation(results not shown).

In the second storage stability test, 6 mutants had been found to beadvantageous. The activity is shown in % of the residual activity of thestarting variant (mutant 18 according to SEQ ID NO:19)—which already hassignificantly improved stability compared to the wild-type enzyme—after4 weeks' storage at 30° C.

Variant SEQ ID NO: Residual activity Mutant 18 19 100% Mutant 19 20 117%Mutant 20 21 118% Mutant 21 22 107% Mutant 22 23 109% Mutant 23 24 109%Mutant 29 117%

It can be seen that all the mutants, without the addition of boric acid,exhibit improved stability in comparison with the starting mutantaccording to SEQ ID NO:19. All the mutants exhibit a washing performancethat is comparable to the wild type according to SEQ ID NO:1, i.e. theyare at most 10% worse in terms of washing performance, which is withinthe measurement fluctuation (results not shown).

In another round of testing, additional mutants were tested forstability, with incubation and storage taking place as described above,but at 40° C.

The following mutants have been found to be advantageous. The activityis shown as % of the residual activity with respect to the initialvalue. The starting variant (mutant 18 according to SEQ ID NO:19)already has markedly improved stability compared to the wild-typeenzyme, and was stored for 4 weeks at 40° C.

Variant SEQ ID NO: Residual activity Mutant 18 19 10% Mutant 24 19%Mutant 25 22% Mutant 26 39% Mutant 27 28% Mutant 28 16%

All the mutants exhibited a further improvement in stability.

The invention claimed is:
 1. A protease comprising an amino acidsequence having at least 85% sequence identity with the amino acidsequence given in SEQ ID NO:1 over its entire length and having, in eachcase based on the numbering according to SEQ ID NO:1, amino acidsubstitutions which occur: (a) at the positions corresponding topositions 9 and 271, and (b) at one or more positions corresponding topositions 29, 48, 101, 130, 131, 133, 144, 224, and 252, in each casebased on the numbering according to SEQ ID NO:1.
 2. The proteaseaccording to claim 1, wherein: (1) the amino acid substitution at theposition 9 is the amino acid substitution 9T, 9H, 9S, or 9A; and whereinthe amino acid substitution at the position 271 is Q271E; (2) the one ormore amino acid substitutions according to (b) is selected from thegroup consisting of A29G, A48V, D101E, N130D, N1305, N130H, G131D,G131N, G131S, G131K, T133K, T133R, T133Y,N144K,N144L,N144A, S224A,S224T, and N252S, in each case based on the numbering according to SEQID NO:1; or (3) both.
 3. The protease according to claim 1, whereinamino acid substitutions occur: (1) at the positions corresponding topositions 9 and 271 and one or more of 130, 131, 133, 144, and 224 ineach case based on the numbering according to SEQ ID NO:1, and (2)optionally at one or more of the positions corresponding to positions29, 48, 101, and 252 in each case based on the numbering according toSEQ ID NO:1.
 4. The protease according to claim 1, wherein the aminoacid substitutions, in each case based on the numbering according to SEQID NO:1: occur at P9T, P9H, P9S, or P9A, corresponding to position 9 andQ271E corresponding to position 271, optionally S216C corresponding toposition 216, as well as additional amino acid substitutions accordingto one or more of the following: (i) N130D, G131S, T133Y, and 5224A;(ii) N1305, G131S, T133Y, N144L, and 5224A; (iii) N130D, G131N, T133K,and N144K; (iv) N130H, G131N, N144K, and 5224A; (v) A29G, N130D, G131N,and T133R; (vi) A29G, D101E, N1305, G131S, S224T, and N252S; (vii)N130H, G131S, and 5224A; (viii) N130D, G131S, T133K, and 5224A; (ix)A29G, D101E, N130D, G131K, and 5224A; (x) N130D, G131N, T133K, N144L,and N252S; (xi) G131S, N144K ,and S224T; (xii) D101E, N130D, G131S,T133Y, N144A, and 5224A; (xiii) A29G, D101E, N1305, S224T, and N252S;(xiv) A48V, G131S, T133R, and 5224A; and (xv) G131D, T133R, and S224A.5. The protease according to claim 1, wherein the amino acidsubstitutions occur, based on the numbering according to SEQ ID NO:1: ata further position corresponding to 166 as 166M, 166Q, or 166A; at oneor more additional positions corresponding to positions 62, 133, 149,156, 172, and 217; or both.
 6. The protease according to claim 5,wherein the amino acid substitutions at the one or more additionalpositions corresponding to positions 62, 133, 149, 156, 172, and 217 areselected from the amino acid substitutions 62S, 133R, 133A, 133K, 1491,156G, 156Q, 172P, 172G, and 217M.
 7. The protease according to claim 1,wherein the protease has an amino acid sequence according to one of SEQID Nos. 3-17 or 19-23.
 8. A protease according to claim 1, furthercomprising a single or multiple conservative amino acid substitution,wherein the protease comprises: (i) the amino acid substitutions P9T,P9H, P9S, or P9A corresponding to position 9 and Q271E corresponding toposition 271 based on the numbering according to SEQ ID NO:1, and (ii)one or more of the amino acid substitutions A29G, A48V, D101E, N130D,N1305, N130H, G131S, G131N, G131D, G131K, T133K, T133R, T133Y, N144K,N144L, N144A, S224A, S224T, and N252S at the positions corresponding topositions 29, 48, 101, 130, 131, 133, 144, 224, and 252 based on thenumbering according to SEQ ID NO:1.
 9. A composition comprising: atleast one protease according to claim 1 and a washing or cleaning agent;and one or more surfactants.
 10. The composition of claim 9, wherein theprotease comprises: (1) the amino acid substitution at the position 9 isthe amino acid substitution 9T, 9H, 9S, or 9A; and wherein the aminoacid substitution at the position 271 is Q271E; (2) the one or moreamino acid substitutions according to (b) is selected from the groupconsisting of A29G, A48V, D101E, N130D, N130S, N130H, G131D, G131N,G131S, G131K, T133K, T133R, T133Y, N144K,N144L,N144A, S224A, S224T, andN252S, in each case based on the numbering according to SEQ ID NO:1; (3)or both.
 11. The composition of claim 9, wherein the protease comprisesthe following amino acid substitutions: (1) at the positionscorresponding to positions 9 and 271 and one or more of 130, 131, 133,144, and 224 in each case based on the numbering according to SEQ IDNO:1, and (2) optionally at one or more of the positions correspondingto positions 29, 48, 101, and 252 in each case based on the numberingaccording to SEQ ID NO:1.
 12. The composition of claim 9, wherein theprotease comprises the following amino acid substitutions, in each casebased on the numbering according to SEQ ID NO:1, which occur at P9T,P9H, P9S, or P9A, corresponding to position 9 and Q271E corresponding toposition 271, optionally S216C corresponding to position 216, as well asadditional amino acid substitutions according to one or more of thefollowing: (i) N130D, G131S, T133Y, and S224A; (ii) N1305, G131S, T133Y,N144L, and S224A; (iii) N130D, G131N, T133K, and N144K; (iv) N130H,G131N, N144K, and S224A; (v) A29G, N130D, G131N, and T133R; (vi) A29G,D101E, N130S, G131S, S224T, and N252S; (vii) N130H, G131S, and S224A;(viii) N130D, G131S, T133K, and S224A; (ix) A29G, D101E, N130D, G131K,and S224A; (x) N130D, G131N, T133K, N144L, and N252S; (xi) G131S, N144K,and S224T; (xii) D101E, N130D, G131S, T133Y, N144A, and S224A; (xiii)A29G, D101E, N130S, S224T, and N252S; (xiv) A48V, G131S, T133R, andS224A; and (xv) G131D, T133R, and S224A.
 13. The composition of claim 9,wherein the amino acid substitutions occur, based on the numberaccording to SEQ ID NO:1: (b1) at the position corresponding to 166 as166M, 166Q, or 166A; (b2) at one or more of the positions correspondingto positions 62, 133, 149, 156, 172, and 217; (b3) or both.
 14. Theprotease according to claim 1, wherein the protease comprises amino acidsubstitutions at the positions corresponding to positions 9 and 271, andat one or more of the positions corresponding to positions 29, 48, 131,133, 144, 224, and 252 in each case based on the numbering according toSEQ ID NO:1.
 15. A protease comprising an amino acid sequence having atleast 85% sequence identity with the amino acid sequence given in SEQ IDNO:1 over its entire length and having, in each case based on thenumbering according to SEQ ID NO:1 amino acid substitutions which occur:(a) at the positions corresponding to positions 9, 130, 144, 252, and271 as the amino acid substitutions 9T, 130D, 144K, 252T, and 271E, and(b) optionally at one or more additional positions corresponding topositions 62, 133, 149, 156, 166, 172, and 217 as amino acidsubstitutions 62S, 133R, 133A, 133K, 1491, 156G, 156Q, 166M, 166Q, 166A,172P, 172G, and 217M.